Process for the sterilization of biological compositions and the product produced thereby

ABSTRACT

The present invention concerns a process for inactivating extracellular and intracellular virus in a biological composition without incurring substantial disruption or inactivation thereof, said process comprising subjecting said composition to a virucidally effective amount of UVA1 irradiation substantially in the absence of UVA2 irradition for a period of time sufficient to thereby inactivate said virus while retaining functionality of said composition. The biological composition is advantageously a product that contains red blood cells or platelets. The process is advantageously carried out in the presence of an irradiation sensitizer compound and/or a quencher. The present invention also concerns the product substantially identical to that produced bythe inventive process.

GOVERNMENT RIGHTS

This work is supported in part by award No. ROI-HL 41221 from theNational Heart, Lung and Blood Institute.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/725,594, filed Oct. 3, 1996, now issued as U.S. Pat. No. 5,789,150,which is, in turn, a divisional of U.S. application Ser. No. 08/344,919,filed Nov. 25, 1994, now issued as U.S. Pat. No. 5,658,722, which is, inturn, a continuation-in-part of U.S. application Ser. No. 08/069,235,filed May 28, 1993, now abandoned, which is, in turn, acontinuation-in-part of U.S. application Ser. No. 08/031,787, filed Mar.15, 1993, now abandoned, which is, in turn, a divisional of U.S.application Ser. No. 07/706,919, filed May 29, 1991, now issued as U.S.Pat. No. 5,232,844, which is in turn a continuation-in-part of U.S.application Ser. No. 07/524,208, filed May 15, 1990, now issued as U.S.Pat. No. 5,120,649.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a process for rendering a biologicalcomposition substantially free of enveloped and non-enveloped virusescontained therein without substantial disruption or inactivation ofcells contained therein and without significant loss of labile proteinsor other valuable biological components also contained therein.

2. Description of Related Art

The problems associated with the application of virucidal procedures tobiological compositions and the efforts to date to overcome theseproblems, including the application of light and chemical agents isreviewed briefly in U.S. Pat. Nos. 5,232,844 and 5,120,649, thedisclosures of which are incorporated herein by reference. See column 1,line 26, through column 4, line 43, of U.S. Pat. No. 5,232,844 andcolumn 1, line 27, through column 4, line 41, of U.S. Pat. No.5,120,649.

Various photodynamic sterilization techniques have been evaluated forinactivating viruses in cellular components of blood. Although many ofthese appear promising for the treatment of red cell concentrates(Matthews et al., "Photodynamic therapy of viral contaminants withpotential for blood banking applications", in Transfusion, 28:81-83(1988); O'Brien et al., "Evaluation of merocyanine 540-sensitizedphotoirradiation as a means to inactivate enveloped viruses in bloodproducts", in J. Lab. Clin. Med., 116:439-47 (1990); and Horowitz etal., "Inactivation of viruses in blood with aluminum phthalocyaninederivatives", in Transfusion, 31:102-8 (1991)), photodynamic viralinactivation methods involving solely oxygen dependent reactions have sofar proved inappropriate for the treatment of platelet concentrates(Proudouz et al., "Inhibition by albumin of merocyanine 540-mediatedphotosensitization of platelets and viruses", in Transfusion, 31:415-22(1991), Dodd et al., "Inactivation of viruses in platelet suspensionsthat retain their in vitro characteristics: comparison ofpsoralen-ultraviolet A and merocyanine 540-visible light methods", inTransfusion, 31:483-90 (1991); and Horowitz et al., "Inactivation ofviruses in red cell and platelet concentrates with aluminumphthalocyanine (AIPc) sulfonates", in Blood Cells, 18:141-50 (1992)).

The use of psoralens together with UVA has demonstrated promise as ameans of photoinactivating viral contaminants in platelet concentrates,although in most studies (Lin et al., "Use of 8-methoxypsoralen andlong-wavelength ultraviolet radiation for decontamination of plateletconcentrates", in Blood, 74:517-525 (1989); and Dodd et al., supra,aminomethyl-trimethylpsoralen (AMT)), the combination of high levels ofvirus inactivation and the maintenance of platelet function werepossible only when air was exchanged with nitrogen prior to UVAirradiation, a cumbersome procedure with inherent variability.

However, it was recently demonstrated (Margolis-Nunno et al., "VirusSterilization in Platelet Concentrates with Psoralen and UVA in thePresence of Quenchers" Transfusion, 22:541-547 (1992)), that for theinactivation of ≧6.0 log₁₀ cell-free vesicular stomatitis virus (VSV) byAMT and UVA, the need for oxygen depletion as a means of protectingplatelets could be obviated by inclusion of mannitol, a scavenger(quencher) of free radicals. (The addition of quenchers of type I (freeradical mediated) or of type II (singlet oxygen mediated) photodynamicreactions is frequently used in other contexts to distinguish whichactive oxygen species produces a particular photodynamic effect.) Underthe conditions used in that study, i.e., 25 μg/ml AMT and 30 minutes ofUVA with 2 mM mannitol, the inactivation of cell-free VSV in air was inpart oxygen dependent since equivalent virus kill (≧6.0 log₁₀) withoxygen depleted required 3 to 4 times more UVA irradiation time (90minutes to 2 hours).

However, while these methods achieved a high level of kill of cell-freelipid enveloped viruses, non-enveloped viruses and latent activelyreplicating and cell-associated viruses were not killed to a high extentunder the conditions reported therein. Therefore, there was the need toeffect the kill of these latter virus forms without causing significantdamage to the desired, valuable components in the biological mixture.Conditions which result in the kill of ≧10⁶ infectious doses of latentor non-enveloped virus have been shown to modify red blood cells andplatelets and result in compromised recovery of labile proteins such asfactor VIII.

In our copending application Ser. No. 08/069,235, the entire contents ofwhich are hereby incorporated by reference, we demonstrated thatsuperior viral inactivation could be achieved at the same time thatsuperior protection of cells and labile proteins was also achieved bysubjecting the biological composition, e.g., platelet concentrates to avirucidally effective amount of irradiation in the presence of (a) amixture of a compound that quenches type I photodynamic reactions and acompound that quenches type II photodynamic reactions or (b) abifunctional compound that is capable of quenching both type I and typeII reactions for a period of time sufficient to inactivate any viruscontained therein.

In spite of these advances, there continues to be a need for novelmethods that achieve an even higher level of kill of both enveloped andnon-enveloped viruses without significant loss of labile proteins orother valuable biological components.

SUMMARY OF THE INVENTION

The overall objective of the present invention was to achieve a highlevel of inactivation of both enveloped and non-enveloped viruses inbiological compositions without incurring substantial disruption orinactivation of cells meant to be contained therein and withoutsignificant loss of labile proteins or other valuable biologicalcomponents also contained therein. This objective was satisfied with thepresent invention, which relates generally to a process for inactivatingextracellular and intracellular virus in a biological compositionwithout incurring substantial disruption or inactivation thereof, saidprocess comprising subjecting said composition to a virucidallyeffective amount of UVA1 irradiation alone or in the presence of anirradiation sensitizer compound for a period of time sufficient toinactivate any virus contained in said composition while retainingfunctionality of said composition. The inventive process can, thus, beused to inactivate viruses in whole blood, red blood cell concentratesand platelet concentrates, without adversely affecting red blood cell orplatelet structure or function. Similarly, the inventive process can beused to inactivate viruses in biological compositions without incurringsubstantial inactivation of desired, soluble biological substances(e.g., coagulation factor concentrates, hemoglobin solutions) containedtherein.

We have found that relatively more damage to cells and labile proteinsis caused by shorter UVA wavelengths (<350 nm) and, therefore, cells andlabile proteins can be better protected by using lamps that do not emitthese shorter wave lengths or filters that eliminate these shorter wavelengths, yet viral inactivation levels are not compromised.

In accordance with another aspect of the invention, the inventiveprocess is advantageously carried out in the presence of quenchercompound. The quencher compound will be capable of quenching type I ortype II photodynamic reactions or both, but preference is given to theuse of (a) mixtures of a compound that quenches type I photodynamicreactions with a compound that quenches type II photodynamic reactionsor (b) bifunctional compounds capable of quenching both type I and typeII reactions.

In accordance with still another aspect of the invention, the inventiveprocess is advantageously combined with a different virucidal method toenhance virus inactivation.

UV treatment alone of either plasma or AHF concentrates results in arelatively high loss of coagulation factor activity under conditionswhich kill ≧10⁵ ID₅₀ of virus; however, it has been discovered that thisloss is significantly reduced (i.e., the recovery is high) whenquenchers of photodynamic reactions are added prior to UV treatment.Compare, Murray et al., "Effect of ultraviolet radiation on theinfectivity of icterogenic plasma", in JAMA, 157:8-14 (1955); and, morerecently, Kallenbach et al., "Inactivation of viruses by ultravioletlight" in Morgenthaler J-J ed. "Virus inactivation in plasma products",in Cum stud Hematol Blood Transfus., 56:70-82 (1989). Thus, the combinedtreatment according to the present invention results in a very highlevel of virus kill while coagulation factor activity is retained athigh levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises two graphs depicting the effect of UVA wavelengthspectrum on psoralen and UVA (PUVA) treated PCs: Treatment of plateletconcentrates (PCs) was with 50 μg/ml AMT, 0.35 mM rutin and indicateddose of irradiation using a transilluminator (Spectroline; Model TR365A) equipped with six 15 watt fluorescent tubes (BLEIT151;Spectroline, Westbury N.Y.) for UVA (320-400 nM; open symbols; 7mW/cm²), and adding a cut-off filter (WG345, Schott Glass Inc., DuryeaPa.) for UVA1 (345-400 nm; filled symbols; 3.5 mW/cm²). FIG. 1a depictsthe rate of platelet aggregation in response to 20 μg/ml collagen asmeasured after overnight storage following treatment and compared tothat of untreated control. FIG. 1b depicts the results when cell-freeVSV was added prior to treatment and infectivity measured after PUVAtreatment.

FIG. 2 is a graph depicting the effect of UVA wavelength on treatment inthe absence of quenchers: PCs were treated with 50 μg/ml AMT and UVA andUVA1 as in FIG. 1 in the absence of rutin. The rate of plateletaggregation in response to collagen was measured as in FIG. 1.

FIG. 3 is a graph depicting the effects of UVA irradiance and wavelengthon function of PUVA treated PCs: PCs treated with UVA (320-400 nm) at 7mW/cm² (open triangle) or UVA1 (345-400 nm) at 3.5 mW/cm² (filledtriangle) as in FIG. 1, or with 7 mW/cm² of 360-370 nm (open circle)(UVP Model #B100A, Thomas Scientific, Swedesboro N.J.) from a mercuryflood bulb UVA light source (UVP).

FIG. 4 comprises two graphs depicting the effects of irradiance (fluencerate) on virus kill (FIG. 4b) and platelet function (FIG. 4a): Treatmentwas with 50 μg/ml AMT, 0.35 mM rutin and UVA (320-400 nm; as in FIG. 1)at two different irradiances (i.e., 7 mW/cm² open squares or 3.5 mW/cm²filled squares). Irradiance was lowered by increasing the distancebetween sample and lamp.

DETAILED DESCRIPTION OF THE INVENTION

Details regarding the make-up of blood, the usefulness of bloodtransfusions, cell-types found in blood and proteins found in blood areset forth at column 6, lines 8-51, of U.S. Pat. No. 5,232,844.Techniques regarding blood plasma fractionation are generally well knownto those of ordinary skill in the art and an excellent survey of bloodfractionation also appears in Kirk-Other's Encylopedia of ChemicalTechnology, Third Edition, Interscience Publishers, Volume 4, pages 25to 62, the entire contents of which are incorporated by referenceherein.

The present invention is directed to subjecting a biological compositionsuch as whole blood, red blood cell concentrates, platelet concentrates,platelet extracts, leukocyte concentrates, semen, ascites fluid, milk,lymphatic fluid, hybridoma cell lines and products derived from any ofthe above, to UVA irradiation alone or in the presence of an irradiationsensitizer compound and, optionally in the presence of a quencher or amixture of quenchers.

The terms "cell-containing composition", "biological composition", or"biological fluid", as used herein, are not to be construed to includeany living organism. Instead, the inventive method is intended to becarried out in an in vitro environment and the cell-containingcomposition, biological composition, or biological fluid obtained by theinventive method will, therefore, be an in vitro produced product, butwill be usable in vivo.

The present invention can be employed to treat the product of acomposition containing non-blood normal or cancerous cells or theproduct of gene splicing.

The term "UVA1" irradiation, as used herein, is intended to mean UVAlight of a wave length ranging from about 340 to 400 nm. The term "UVA",as used herein, is ordinarily intended to refer to UVA light having abroader (320-400 nm) emission spectrum, i.e., including so-called "UVA2"irradiation, which is by convention UVA light of a wavelength rangingfrom about 320 to 340 nm.

Details on the application of UVA (or UVA1) radiation to effect virusinactivation are well known to those skilled in the art. Typicalradiation fluences range for the invention are approximately 0.5-100J/cm² (preferably 1-50 J/cm²).

When utilized, suitable quenchers are any substances known to react withboth free radicals (so-called "type I quenchers") and reactive forms ofoxygen (so-called "type II quenchers"). Representative quenchers includeunsaturated fatty acids, reduced sugars, cholesterol, indolederivatives, and the like, azides, such as sodium azide, tryptophan,polyhydric alcohols such as glycerol and mannitol, thiols such asglutathione, superoxide dismutase, flavonoids, such as quercetin andrutin, amino acids, DABCO, vitamins such as vitamin A, C and E and thelike.

The quencher is used in conventional quenching amounts, but,surprisingly, when used, the overall process results in preferentialdamage to the virus but not to the desired biological material.

In accordance with the present invention, superior virus kill isachieved by quenching both type I and type II photodynamic reactions,i.e., by using a mixture of type I and type II quenchers or by usingcompounds, e.g., flavonoids, that are known to quench both type I andtype II reactions. The range of virus kill is in most cases broader thanthat achieved by using type I or type II quenchers alone--even ascompared to increased concentrations of the type I or type IIquencher--or by using mixtures of type I quenchers or mixtures of typeII quenchers. Moreover, this broader range of virus kill is achievedwithout sacrificing intact cell functionality or structure.

The inventive process is typically carried out over a temperature rangeof 0-42° C., preferentially 15-37° C. and most preferentially 15-25° C.The inventive process is typically carried out at pH 6.5-8, mostpreferentially 7.2-7.6. Samples are typically subjected to the inventiveprocess for a period of time of less than 24 hours. Samples can also betreated frozen.

In an embodiment of the present invention, the biological composition issubjected to irradiation and the quencher or quencher mixture in thepresence of an irradiation sensitizer. In this context, suitableirradiation sensitizer compounds for use in the present inventioninclude phthalocyanines, purpurins, and other molecules which resemblethe porphyrins in structure (as described above) as well as photoactivecompounds excited by ultraviolet light (e.g., psoralen,8-methoxypsoralen, 4'-aminomethyl-4,5',8-trimethyl psoralen, bergapten,and angelicin), dyes which absorb light in the visible spectrum (e.g.,hypericin, methylene blue, eosin, fluoresceins and flavins), and dyeswhich absorb X-irradiation (e.g. brominated psoralen, brominatedhematoporphyrin, iodinated phthalocyanine) . The use of such irradiationsensitizers would be readily apparent to those skilled in the art and ispreferably substantially as described in U.S. Pat. No. 5,120,649 andU.S. Ser. No. 07/706,919, filed May 29, 1991, the entire disclosures ofwhich are incorporated herein by reference.

According to another embodiment of the invention, the treatment of thebiological composition with irradiation and quencher or quencher mixtureis combined with a second virucidal method. This second virucidal methodcan be any method used conventionally to inactivate enveloped and/ornon-enveloped viruses such as, merely for example, heat treatment, dryor otherwise, pH manipulation, treatment with lipid solvents and/ordetergents, a separate irradiation treatment, e.g., withgamma-irradiation, or treatment with chemical agents, e.g.,formaldehyde.

Non-limiting examples of lipid coated, human pathogenic viruses that canbe inactivated by the present invention include vesicular stomatitisvirus (VSV), Moloney sarcoma virus, Sindbis virus, humanimmunodeficiency viruses (HIV-1; HIV-2), human T-cell lymphotorophicvirus-I (HTLV-I), hepatitis B virus, non-A, non-B hepatitis virus (NANB)(hepatitis C), cytomegalovirus, Epstein Barr viruses, lactatedehydrogenase elevating virus, herpes group viruses, rhabdoviruses,leukoviruses, myxoviruses, alphaviruses, Arboviruses (group B),paramyxoviruses, arenaviruses and coronaviruses. Non-limiting examplesof non-enveloped viruses that can be inactivated by the presentinvention include parvovirus, polio virus, hepatitis A virus, entericnon-A, non-B hepatitis virus, bacteriophage M13 and satelliteadeno-associated virus (AAV).

Cell-containing compositions to be treated according to the inventionhave ≧1×10⁸ cells/ml, preferably ≧1×10⁹ cells/ml and most preferably≧1×10¹⁰ cells/ml. Furthermore, cell-containing compositions to betreated according to the invention have preferably >4 mg/ml protein andmore preferably >25 mg/ml protein and most preferably 50 to 60 mg/mlprotein (unwashed cells).

Non-cell containing compositions to be treated according to theinvention have ≧0.1 mg/ml and preferably ≧5 mg/ml protein.

In the inventive process, at least 10⁴, preferably 10⁶, infectious unitsof virus parasite or other pathogen are inactivated.

The biological compositions treated according to the invention, whileinitially containing ≧1000 infectious units of virus/L, after the virushas been inactivated and treatment according to the invention has beenconducted, have, in the case of cell-containing compositions, aretention of intact cell functionality and structure of greater than70%, preferably greater than 80% and most preferably greater than 95%.In the case of biological fluids, a retention of biological activity ofgreater than 75%, preferably greater than 85%, and most preferablygreater than 95% can be achieved.

By the inactivation procedure of the invention, most if not virtuallyall of the viruses contained therein would be inactivated. A method fordetermining infectivity levels by inoculation into chimpanzees (in vivo)is discussed by Prince, A. M., Stephen, W., Bortman, B. and van denEnde, M. C., "Evaluation of the Effect of Beta-propiolactone/UltravioletIrradiation (BPL/UV) Treatment of Source Plasma on HepatitisTransmission by Factor IX Complex in Chimpanzees", Thrombosis andHemostasis, 44: 138-142, (1980).

According to the invention, inactivation of virus is obtained to theextent of at least "4 logs", preferably ≧6 logs, i.e., virus in thesample is totally inactivated to the extent determined by infectivitystudies where that virus is present in the untreated sample in such aconcentration that even after dilution to 10⁴ (or 10⁶), viral activitycan be measured.

The present invention describes inactivating viruses, whilesimultaneously retaining labile blood cell functional and structuralfeatures.

Functional activities of red cells are ascertained by measurements ofmetabolite levels, enzymatic activities, electrolyte levels and oxygencarrying capacity. Structural integrity of red cells is assessed bymeasurements of hemoglobin release, osmotic fragility, survival in vivofollowing radiolabeling with chromium-51, antigenicity and by evaluationof modification of cell surface proteins. Further evidence of theintegrity of treated red blood cells comes from the measurement of theircirculatory half-life.

While those skilled in the art will appreciate that the inventiveprocess will be useful to sterilize most blood products, including, butnot limited to, whole blood, red blood cell concentrates, plateletconcentrates, platelet extracts, leukocyte extracts, blood proteinconcentrates, etc., the inventive method will prove especially valuablefor the sterilization of platelet concentrates and platelet extracts. Asalluded to previously, there is a special need for sterilizationprocesses that afford superior viral inactivation while at the same timebeing protective of platelets. The present invention satisfies thisneed.

Functional activities of platelets are determined by their ability toaggregate in the presence of certain biological agents and theirmorphology and, also, by assessing the maintenance of the pH uponlimited storage of a solution containing the platelets and in vivohemostatic effectiveness using the rabbit ear bleeding time technique(Wagner et al., 1993, Blood, 82:3489. Structural integrity of plateletsis assessed by in vivo survival following radiolabeling with indium-111and identification of the presence of specific platelet antigens.

After treatment with the photoreactive compound, excess photoreactivecompound can be removed by centrifugation, washing dialysis, and/oradsorption onto hydrophobic matrices.

In an embodiment of the present invention, the treated cell-containingfraction from the inventive process is transfused or returned to thedonor, e.g., human donor, from which the initial cell-containingfraction was derived. In this manner, the level of circulating virus inthe donor will be reduced, thus improving the donor's ability to clearvirus and/or improving the efficacy of antiviral drugs.

Factor VIII and factor IX coagulant activities are assayed bydetermining the degree of correction in APTT time of factor VIII--andfactor IX--deficient plasma, respectively. J. G. Lenahan, Philips andPhilips, Clin. Chem., Vol. 12, page 269 (1966).

The activity of proteins which are enzymes is determined by measuringtheir enzymatic activity. Factor IX's activity can be measured by thattechnique.

Binding proteins can have their activities measured by determining theirkinetics and affinity of binding to their natural substrates.

Lymphokine activity is measured biologically in cell systems, typicallyby assaying their biological activity in cell cultures.

Protein activity generally is determined by the known and standard modesfor determining the activity of the protein or type of protein involved.

The invention will now be described with reference to the followingnon-limiting examples.

EXAMPLES

Materials and Methods

Platelet Concentrates (PCs)

PCs, released after routine blood bank testing, were typically 24 to 48hours old when treated. Prior to treatment, the PCs were stored at 22 to24° C. in the bags (PL 732, Fenwal Laboratories, Deerfield, Ill.) inwhich they were received and constantly agitated on a platelet rotator(Helmer Labs, St. Paul, Minn.).

Psoralen Solutions

4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was purchased from HRIAssoc. Inc., Concord, Calif. Stock solutions of AMT (4 mg/ml) wereprepared in distilled water.

Model Virus Studies

The inactivation of vesicular stomatitis virus (VSV), a lipid enveloped,RNA virus was studied.

VSV was cultured in human A549 cells. Culturing and assay procedureswere similar to those described in Horowitz, B., Wiebe, M. E., Lippin,A. and Stryker, M. H., "Inactivation of Viruses in Labile BloodDerivatives", Transfusion, 1985, 25:516-522. Infectivity of VSV wasassessed by endpoint, 10-fold serial dilutions in DMEM culture medium(Gibco Laboratories, Grand Island, N.Y.) with 10% fetal calf serum (FCS;MA Bioproducts, Walkersville, Md.). Each dilution was used to inoculateeight replicate wells of human A549 cells in 96-well microtiter plates.Virus-induced cytopathology was scored after 72 hours of incubation at37° C. in 5% CO₂. The reported virus titer was calculated using theSpearman-Karber method (Spearman, C., "The Method of Right and WrongCases' (`Constant Stimuli`) Without Gauss's Formula", Br. J. Psychol.,1908;2:227-242) and indicates the quantity of virus which infects 50% ofthe tissue culture wells (TCID₅₀).

For assessment of platelet function, measurement was made of the rate ofplatelet aggregation (% control) in response to the addition of 20 μm/mlof collagen.

For assessment of virus inactivation, the virucidal reaction was stoppedby 10-fold dilution into DMEM containing 5% fetal calf serum, and thecells when present were removed by centrifugation at 1500 rpm for 10minutes. The lack of virus inactivation at this dilution or in theabsence of irradiation was confirmed for each of the inactivationconditions studied. Samples were sterile filtered (Swinnex filters,Millipore Corp., Bedford, Mass.) and frozen at -70° C. or below untilassay.

RESULTS

In order to assess the effect on viral specificity of PUVA treatments,i.e., viral kill versus platelet function, we compared the effects ofdifferent UVA lamps and filters on identical samples under otherwisesimilar viral inactivation conditions (e.g., 50 μg/ml AMT, 0.35 mM rutinand 39 J/cm²). In one set of experiments, a fluorescent lamp with abroader UVA emission spectrum ranging over 320-400 nm (UVA) was used,whereas in the second set of experiments, there was used a filter whichcut-off wavelengths below 345 nm.

The results show that, while virus kill was equivalent with equal dosesof UVA1 or UVA, platelet function surprisingly was significantly bettermaintained with UVA1 than with UVA (see FIG. 1). This was also true forPUVA treatment in the absence of rutin or other quenchers (see FIG. 2)where the use of a filter had a positive effect on platelet aggregationafter treatment.

The improvement in platelet aggregation response when shorterwavelengths were excluded during PUVA treatment (see FIG. 3) was notjust an effect of a lower fluence rate (see FIG. 4). With a UVA1 sourcehaving a steep emission peak near 365 nm (UVA 365) and a high (6.7mW/cm²) irradiance results were similar to those with UVA1 at a lowirradiance (3.5 m W/cm²) (FIG. 3). Maintenance of platelet function wasdependent on the spectral emission of the UVA radiation source (FIG. 3)rather than on its intensity (FIG. 4).

Collectively, these data show that relatively more damage to plateletsis caused by the shorter UVA wavelength range (<345 nm), and that UVAdose appropriate for PC treatment is therefore somewhat dependent onirradiator emmission spectrum. They also suggest that virus specificityof PUVA treatment of PCs can be enhanced by removal of UVA wavelengthsbelow 345 nm.

It will be appreciated that the instant specification is set forth byway of illustration and not limitation, and that various modificationsand changes may be made without departing from the spirit and scope ofthe present invention.

What is claimed is:
 1. A process for treating a biological compositionto inactivate an extracellular or intracellular virus that may bepresent therein, said process comprising performing two viralinactivation methods on said biological composition, wherein one of saidtwo methods comprises the steps of: adding to the biological compositionan irradiation sensitizer and then subjecting the resultant compositionto a virucidally effective amount of UVA1 irradiation in the absence ofUVA2 radiation and wherein the second viral inactivation method isselected from the group consisting of treatment with a solvent and adetergent, and a heat treatment.
 2. The process according to claim 1,wherein the irradiation sensitizer is a psoralen.
 3. The processaccording to claim 2, wherein the psoralen is4'-aminomethyl-4,5',8-trimethyl psoralen.
 4. The process according toclaim 1, wherein the biological composition contains red blood cells orplatelets.
 5. The process according to claim 4, wherein the irradiationsensitizer is a psoralen.
 6. The process according to claim 5, whereinthe psoralen is 4'-aminomethyl-4,5',8-trimethyl psoralen.
 7. The processaccording to claim 1, wherein a quencher is added to said biologicalcomposition before said biological composition is subjected to said UVA1irradiation.
 8. The process according to claim 7, wherein said quencheris selected from the group consisting of (a) a mixture of one or morecompounds that quench type I photodynamic reactions and one or morecompounds that quench type II photodynamic reactions or (b) abifunctional compound which quenches both type I and type II reactionsor (c) a mixture of a bifunctional compound that is capable of quenchingboth type I and type II reactions and an additional quencher whichquenches either type I, type II or both type I and type II reactions. 9.The process according to claim 8, wherein the quencher is a bifunctionalcompound.
 10. The process according to claim 9, wherein the bifunctionalcompound is selected from the group consisting of quercetin, chrysin,cachetin, rutin, hesperidin and naringen.
 11. The process according toclaim 10, wherein the biological composition contains red blood cells orplatelets.
 12. The process according to claim 1, wherein the other viralinactivation method comprises treatment with a solvent and a detergent.13. The process according to claim 1, wherein the other viralinactivation method comprises heat treatment.